Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions of CD64/CD28/CD3ζ in colorectal cancer (CRC) and squamous cell carcinoma of the head and neck.
The CD64/CD28/CD3ζ -SFG retroviral vector was used to produce viruses for T-cell transduction. T-cell expansion and differentiation were monitored via flow cytometry. Gene expression was assessed by RNA-seq. Bioenergetics were documented on a Seahorse extracellular flux analyzer. CD64/CD28/CD3ζ polarization was identified via confocal microscopy. Cytotoxicity was determined by MTT assay and bioluminescent imaging, and flow cytometry. Tridimensional antitumor activity of CD64/CD28/CD3ζ T cells was achieved by utilizing HCT116-GFP 3D spheroids via the IncuCyte S3 Live-Cell Analysis system. The intraperitoneal distribution and antitumor activity of NIR-CD64/CD28/CD3ζ and NIR-nontransduced T cells were investigated in CB17-SCID mice bearing subcutaneous FaDu Luc + cells by bioluminescent and fluorescent imaging. IFNγ was assessed by ELISA.
Compared to CD16/CD8a/CD28/CD3ζ T cells, CD32/CD8a/CD28/CD3ζ T cells, and non-transduced T cells, CD64/CD28/CD3ζ T cells exhibited the highest levels of cell expansion and persistence capacity. A total of 235 genes linked to cell division and 52 genes related to glycolysis were overexpressed. The glycolytic phenotype was confirmed by functional in vitro studies accompanied by preferential T-cell effector memory differentiation. Interestingly, oxamic acid was found to inhibit CD64-CR T cell proliferation, indicating the involvement of lactate. Upon CD64/CD28/CD3ζ T-cell conjugation with CRC cells, CD64/CD28/CD3ζ cells polarize at immunological synapses, leading to CRC cell death. CD64/CD28/CD3ζ T cells kill SCCHN cells, and in combination with the anti-B7-H3 mAb (376.96) or anti-EGFR mAb, these cells trigger antibody-dependent cellular cytotoxicity (ADCC) in vitro under 2D and 3D conditions. The 376.96 mAb combined with CD64/CD28/CD3ζ T cells had anti-SCCHN activity in vivo. In addition, they induce the upregulation of PD-L1 and HLA-DR expression in cancer cells via IFNγ. PD-L1 positive SCCHN cells in combination with anti-PD-L1 mAb and CD64-CR T cells were killed by ADCC, which enhanced direct cytotoxicity. These findings indicate that the glycolytic phenotype is involved in CD64-CR T cell proliferation/expansion. These cells mediate long-lasting HLA-independent cytotoxicity and ADCC in CRC and SCCHN cells.
CD64/CD28/CD3ζ T cells could significantly impact the rational design of personalized studies to treat CRC and SCCHN and the identification of novel FcγR ALs in cancer and healthy cells.
© 2025. The Author(s).

Abstract Background Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions of CD64/CD28/CD3ζ in colorectal cancer (CRC) and squamous cell carcinoma of the head and neck. Methods The CD64/CD28/CD3ζ -SFG retroviral vector was used to produce viruses for T-cell transduction. T-cell expansion and differentiation were monitored via flow cytometry. Gene expression was assessed by RNA-seq. Bioenergetics were documented on a Seahorse extracellular flux analyzer. CD64/CD28/CD3ζ polarization was identified via confocal microscopy. Cytotoxicity was determined by MTT assay and bioluminescent imaging. Tridimensional antitumor activity of CD64/CD28/CD3ζ T cells was achieved by utilizing HCT116-GFP 3-D spheroids via the IncuCyte S3 Live-Cell Analysis system. The intraperitoneal distribution and antitumor activity of NIR-CD64/CD28/CD3ζ and NIR-nontransduced T cells were investigated in CB17-SCID mice bearing subcutaneous FaDu Luc + cells by bioluminescent and fluorescent imaging. IFNγ was assessed by ELISA. Results Compared to CD16/CD8a/CD28/CD3ζ T cells, CD32/CD8a/CD28/CD3ζ T cells, and nontransduced T cells, CD64/CD28/CD3ζ T cells exhibited the highest levels of cell expansion and persistence capacity. A total of 235 genes linked to cell division and 52 genes related to glycolysis were overexpressed. The glycolytic phenotype was confirmed by functional in vitro studies accompanied by preferential T-cell effector memory differentiation. Upon CD64/CD28/CD3ζ T-cell conjugation with CRC cells, CD64/CD28/CD3ζ cells polarize at immunological synapses, leading to CRC cell death. CD64/CD28/CD3ζ T cells kill SCCHN cells, and in combination with the anti-B7-H3 mAb (376.96) or anti-EGFR mAb, these cells trigger ADCC in vitro under 2D and 3D conditions. The 376.96 mAb combined with CD64/CD28/CD3ζ T cells had anti-SCCHN activity in vivo. In addition, they induce the upregulation of PD-L1 and HLA-DR expression on cancer cells via IFNγ. PD-L1 upregulation resulted in the generation of ADCC, which enhanced direct cytotoxicity. These findings indicate that, despite the glycolytic phenotype, these cells mediate long-lasting HLA-independent cytotoxicity and ADCC in CRC and SCCHN cells. Conclusions CD64/CD28/CD3ζ T cells could significantly impact the rational design of personalized studies to treat CRC and HNSCC and the identification of novel FcγR ALs in cancer and healthy cells.

Blood phagocytes, such as neutrophils and monocytes, generate reactive oxygen species (ROS) as a part of host defense response against infections. We investigated the mechanism of Fcγ-Receptor (FcγR) mediated ROS production in these cells to understand how they contribute to anti-malarial immunity. Plasmodium falciparum merozoites opsonized with naturally occurring IgG triggered both intracellular and extracellular ROS generation in blood phagocytes, with neutrophils being the main contributors. Using specific inhibitors, we show that both FcγRIIIB and FcγRIIA acted synergistically to induce ROS production in neutrophils, and that NADPH oxidase 2 and the PI3K intracellular signal transduction pathway were involved in this process. High levels of neutrophil ROS were also associated with protection against febrile malaria in two geographically diverse malaria endemic regions from Ghana and India, stressing the importance of the cooperation between anti-malarial IgG and neutrophils in triggering ROS-mediated parasite killing as a mechanism for naturally acquired immunity against malaria.
© 2023. The Author(s).

The difficulty to unambiguously identify the various subsets of mononuclear phagocytes (MNPs) of the intestinal lamina propria has hindered our understanding of the initial events occurring after mucosal exposure to HIV-1. Here, we compared the composition and function of MNP subsets at steady-state and following ex vivo and in vivo viral exposure in human and macaque colorectal tissues. Combined evaluation of CD11c, CD64, CD103, and CX3CR1 expression allowed to differentiate lamina propria MNPs subsets common to both species. Among them, CD11c+ CX3CR1+ cells expressing CCR5 migrated inside the epithelium following ex vivo and in vivo exposure of colonic tissue to HIV-1 or SIV. In addition, the predominant population of CX3CR1high macrophages present at steady-state partially shifted to CX3CR1low macrophages as early as three days following in vivo SIV rectal challenge of macaques. Our analysis identifies CX3CR1+ MNPs as novel players in the early events of HIV-1 and SIV colorectal transmission.
© 2022 The Author(s).

It is unknown whether antibody-mediated enhancement (ADE) contributes to the pathogenesis of COVID-19, and the conditions for ADE needs to be elucidated. We demonstrated that without inducing an ACE2-independent ADE on Raji cells, the neutralizing antibody CB6, a mouse anti-S1 serum and convalescent plasma, induced ADE on cells expressing FcγRIIA/CD32A and low levels of endogenous ACE2. ADE occurred at sub-neutralizing antibody concentrations, indicating that unneutralized S protein was required for ADE. The enhanced infectivity of 614G variant was higher than that of 614D wildtype in the presence of antibodies, further suggesting that ADE may be influenced by virus strains with different ACE2-binding affinity. Finally, knockdown of ACE2 or treatment with a fusion-inhibition peptide EK1C4 significantly reduced ADE. In conclusion, we identified an ADE mechanism mediated by neutralizing antibodies against SARS-CoV-2. ACE2 may act as a secondary receptor required for the antibody- and FcγR-mediated enhanced entry of SARS-CoV-2.
© 2021 The Author(s).